‘Qualitative

‘Qualitative
Test for
Test for
Secondary
Secondary
Metabolites’
Metabolites’
By: Keiko A. Rodrigo
What are secondary
metabolites?
 Metabolites
➔ A metabolite is a product of metabolism
◆ It is the sum of chemical reactions involved in maintaining the living state
of cells and the organism.

➔ Two types of plant metabolites

Primary Metabolites Secondary Metabolites

- Essential for growth phase - Not involved the growth

- Role: maintenance, growth, phase
development. (Needed for - Role: protection, signaling,
survival) defense
Secondary Metabolites
➔ Their role is to defend the organism from environmental
stressors such as:
◆ Predators
◆ Competition
◆ Disease
◆ Any other type of interaction among other organisms
➔ They are produced by plants, fungi, and bacteria and play an
important role in plant defense. They could be called the
immune system of the plant
➔ One of the most known secondary metabolites is caffeine.
◆ It is one of the active compounds of the coffee tree and its
role is to simply defend. When coffee leaves fall on the
ground, they contaminate the soil with caffeine, which
makes it difficult for other plants to germinate. Caffeine
can also be toxic for many insects and herbivores.
What is Qualitative
Testing?
Qualitative Testing
➔ Qualitative testing is used
to
◆ Observe and
understand small
details.
◆ Measure the presence
or absence of a
substance
◆ Evaluate certain
characteristics
 What does qualitative
testing have to do with
secondary metabolites?
What is its purpose?
➔ The tests are usually performed to determine
the presence or absence of secondary
metabolites in plants.
➔ Humans utilize the secondary metabolites to
create
◆ Medicines
◆ Flavorings
◆ Pigments
◆ Recreational Drugs
Example: Erythromycin tablets

➔ Most of these products are derived from plants since the biological effects of plant
secondary metabolites on humans have been known since ancient times. And were
used to treat infections, inflammation, cold, digestive problems to pain and many
other health disorders
There are 5 main classifications of secondary
metabolites

Alkaloids Enzyme Nonribosomal

Cofactors Peptides

Terpenoids and Fatty-acid derived

Steroids substances and
polyketides
 6 Classes of Plant Secondary Metabolites

Phenolics Alkaloids Saponins

Terpenes Lipids Carbohydrates


a.) Lead acetate test (For Phenols and
flavonoids)
● Treat the extract with few
drops of lead acetate
solution.
● Formation of yellow
precipitate indicated the
presence of flavonoids.
Phenolics
b.) Alkaline reagent test (For
Flavonoids)
● Treat the extract with few
drops of hydroxide
solution.
● Formation of intense
yellow colour, which
becomes colourless on
further addition of dilute
acid, indicates presence of
flavonoids
c.) Ferric Chloride Test (For Tannins
and Phenols)
● Add a few drops of ferric
chloride solution to the
extract solution
● Development of intense
blue, red, purple or green
colour indication presence
of phenols/flavonoids
 Alkaloids
a.) Hager’s Test b.) Wagner’s Test c.) Dragendroff’’s Test d.) Mayer’s Test

● Filtrate was treated ● Extracts when treated ● Filtrate was treated ● Filtrate was treated
with Hager’s reagent with Wagner’s reagent with Dragendroff’s with Mayer’s Reagent
(Saturated picric acid (iodine in potassium Reagent (potassium (potassium mercuric
solution) iodide) bismuth iodide iodide solution)
● Presence of alkaloids ● Formation of solution) ● Formation of a whitish
were confirmed by brown/reddish ● Formation of orange yellow or cream
formation of yellow precipitate indicates red precipitate colored precipitate
colored precipitate the presence of indicated presence of indicated the presence
alkaloids alkaloids of alkaloids
 Saponins
a.) Foam Test b.) Froth Test

● Small quantity of extract is shaken with ● Buffered distilled water is transferred to a

water vial and shaken vigorously
● If foam persists for ten minutes indicates the ● Formation of 1cm layer of foam indicated
presence of saponins. presence of saponins
Terpenes
a.) Salkowski’s Test

● Extract is mixed in chloroform and

concentrate sulphuric acid was
carefully added to form a layer
● A reddish brown coloration of the
interface was formed to show positive
result for presence of terpenoids.
 Lipids
a.) Spot Test
● Place small quantity of extract in
between two filter papers.
● Oil stain produced with any extract
showed the presence of fixed oils and
fats in the extracts
b.) Saponification test
● Add a few drops of 0.5N alcoholic
potassium hydroxide extract with few
drops of phenolphthalein solution
● Heat on a water bath for 1-2 hours
● Formation of soap indicated the
presence of fixed oils and fats in the
extracts
Carbohydrates
a.) Molisch’s Test

● Filtrate was treated with 2 drops of

alcohol a-naphthol solution in a test
tube, shaken.
● Add cone. Sulphuric acid from the
side of the test tube
● Development of a violet ring at the
junction of two liquid confirmed the
presence of carbohydrates.
To sum things up...
➔ Secondary metabolites are a plant’s immune system
➔ We do qualitative tests for secondary metabolites to create various products such as medicines.
➔ For the five main classification of secondary metabolites, there are seven main classes, with different tests:
◆ Phenolics
● Lead acetate test (For phenols and flavonoids)
● Alkaline reagent test (For Flavonoids)
● Ferric Chloride Test (For Tannins and Phenols)
◆ Alkaloids
● Hager’s Test
● Wagner’s Test
● Dragendroff’s Test
● Mayer’s Test
◆ Saponins
● Foam Test
● Forth Test
◆ Terpenes
● Salkowski’s Test
◆ Lipids (Fixed Oils and fats)
● Spot Test
● Saponification Test
◆ Carbohydrates
● Molisch’s Test